We will test the hypothesis that a unique recombinant fusion protein derived from a cell-penetrating viral capsid shell can transport DNA intercalating drugs through a tiny nucleic acid carrier, and target such drugs to specific tumor cells causing tumor-specific delivery and cell death. To test the feasibility of this new type of therapeutic, we have chosen HER2+ breast cancer as our tumor target. HER2+ breast tumors, which overexpress subunit 2 of the human epidermal growth factor receptor (HER), comprise a significant subset of breast cancers that are recalcitrant to standard methods of treatment, and predict a high mortality. We will take advantage of the rapid endocytic uptake induced by natural ligand-receptor interactions and the membrane penetrating activity of a viral protein to deliver a drug into the cancer cell and induce cytotoxicity from within. The Specific Aims of this project are to test the hypotheses that: 1. Key molecular motifs enhance cell targeting and penetration of a modified capsid protein in vitro and in vivo. A series of variants of the heregulin-targeted protein, HerPBK10, will be generated and tested for targeted cell binding, uptake, intracellular penetration and trafficking on a panel of HER2+ and HER2- breast cancer cell lines and primary cells, and isogenic cell lines expressing different HER subunit levels. Immunofluorescence and confocal microscopy will be used to analyze uptake and trafficking, and subcellular fractionation will be used to confirm and quantify the results. Subcellular markers and trafficking inhibitors will help determine uptake pathways. Finally, the variants will be tested for tumor targeting in vivo. Variants that yield optimal targeting and uptake will be incorporated into the bioconjugate proposed in the subsequent aims. 2. A modified capsid protein assembles with a double-stranded oligonucleotide (ds-oligo) and DNA intercalating agent to form noncovalent bioconjugates that target HER2+ breast cancer cells in vitro. The optimal protein variant selected from Aim 1 will be combined with ds-oligos of varied lengths and DNA intercalating agent to form noncovalent conjugates. We will use UV/Vis absorbance and fluorometry to assess assembly parameters and stability under different storage conditions, and in serum. We will test each assembly for targeted toxicity in separate and mixed HER2+ and HER2- cell cultures using metabolic assay, and receptor specificity will be verified by competitive inhibition with free ligand. Finally, we will investigate the mechanism of targeted toxicity and test our proposed model of drug uptake and intracellular release. These results will dictate the optimal assembly parameters and dosage of bioconjugate to test in Aim 3. 3. An optimized bioconjugate comprised of a modified capsid protein, ds-oligo, and DNA intercalator targets HER2+ tumor cells and imparts therapeutic efficacy in vivo. We will utilize a nude mouse model of HER2+ breast cancer to establish an in vivo dose curve for determining the minimal intratumoral dosage effecting maximal tumor ablation and determine the timeline required for growth inhibition and regression. Biodistribution and pharmacokinetics will be determined by in vivo imaging and measuring conjugate levels in blood, respectively, in treated mice. We will determine the therapeutic efficacy of conjugate delivered systemically and tumor volumes will be measured to determine growth inhibition. Finally, immunogenicity will be determined using absorbance assays of blood taken from treated immune competent mice. PUBLIC HEALTH RELEVANCE: This research project is relevant to public health because it will result in the development of a novel therapeutic that can specifically target HER2+ breast cancer. This targeted therapy should be an improvement over conventional treatment methods because normal cells should not be affected. As HER2+ breast cancer does not respond well to conventional therapies, this alternative therapy could provide a significant contribution to breast cancer treatment, and could be modified to target therapy to other types of cancer.